Amplification d'ADN des génomes multiples pour détecter des délétions

https://www.cell.com/cell/pdf/0092-8674(88)90383-2.pdf

The Complete Sequence of Dystrophin Predicts a Rod-Shaped Cytoskeletal Protein

By means of a selective DNA amplification technique called polymerase chain reaction, proviral sequences of the human immunodeficiency virus (HIV-1) were identified directly in DNA isolated from peripheral blood mononuclear cells (PBMCs) of persons seropositive but not in DNA isolated from PBMCs of persons seronegative for the virus. Primer pairs from multiple regions of the HIV-1 genome were used to achieve maximum sensitivity of provirus detection. HIV-1 sequences were detected in 100% of DNA specimens from seropositive, homosexual men from whom the virus was isolated by coculture, but in none of the DNA specimens from a control group of seronegative, virus culture-negative persons. However, HIV-1 sequences were detected in 64% of DNA specimens from seropositive, virus culture-negative homosexual men. This method of DNA amplification made it possible to obtain results within 3 days, whereas virus isolation takes up to 3 to 4 weeks. The method may therefore be used to complement or replace virus isolation as a routine means of determining HIV-1 infection.

http://science.sciencemag.org/content/sci/239/4837/295.full.pdf

DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells

A sequencing method called genomic amplification with transcript sequencing (GAWTS) is described that is based on amplification with the polymerase chain reaction (PCR). GAWTS bypasses cloning and increases the rate of sequence acquisition by at least fivefold. The method involves the attachment of a phage promoter onto at least one of the PCR primers. The segments amplified by PCR are transcribed to further increase the signal and to provide an abundance of single-stranded template for reverse transcriptase-mediated dideoxy sequencing. An end-labeled reverse transcriptase primer complementary to the desired sequence generates the additional specificity required to generate unambiguous sequence data. GAWTS can be performed on as little as a nanogram of genomic DNA. The rate of GAWTS can be increased by coamplification and cotranscription of multiple regions as illustrated by two regions of the factor IX gene. Since GAWTS lends itself well to automation, further increases in the rate of sequence acquisition can be expected.

http://science.sciencemag.org/content/sci/239/4839/491.full.pdf

Genomic amplification with transcript sequencing.

A 230 kb genomic region from the Duchenne muscular dystrophy gene has been cloned in a cosmid walk, using an improved vector and by screening the same unamplified library for all steps The region cloned surrounds the translocation breakpoint characterized by Worton et al and Ray et al, and overlaps by 70 kb the Pert region cloned by Monaco et al We have identified a region of strong sequence conservation in mammals and chicken, and comparison of the homologous sequences in chicken and man has indicated the presence of two putative protein coding exons Comparison with the sequence recently published by Koenig et al shows that only one is present in the Duchenne cDNA, and this raises the question of the functional significance of the other conserved sequence Single copy probes and whole cosmids generated during this work have been used to analyse the corresponding region in Duchenne patients Of five independant patients shown to be deleted for a probe 30 kb in 3' of the translocation breakpoint, three have the 5' endpoint of the deletion within a region of less than 20 kb, 100 kb away from the probe used to ascertain the deletion This might suggest the presence of a region where deletions occur preferentially

/pdf/a-230kb-cosmid-walk-in-the-duchenne-muscular-dystrophy-gene-15z20fcv6f.pdf

Amplification d'ADN des génomes multiples pour détecter des délétions

References

The Complete Sequence of Dystrophin Predicts a Rod-Shaped Cytoskeletal Protein

DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells

Genomic amplification with transcript sequencing.

A 230kb cosmid walk in the Duchenne muscular dystrophy gene: detection of a conserved sequence and of a possible deletion prone region.